Development Of Divalent Inactivated Vaccine For Mink Enteritis Parvovirus

Mink viral enteritis,also known as leukopenia,is an acute,highly contagious disease caused by mink enteritis virus（MEV）.It has seriously impeded the healthy development of the mink farming industry and caused major economic losses.In recent years,the immune failure of mink virus enteritis has often occurred and is endemic in some areas.In 2016-2017,the epidemiological survey results of our laboratory showed that feline parvovirus（FPV）is prevalent in China’s mink,and FPV and MEV can cause typical scorpion viral enteritis.For this reason,the enteritis parvovirus bivalent inactivated vaccine was developed in the study.The feline parvovirus FPV-SD1 strain（hereinafter referred to as SD1）and the mink enteritis parvovirus MEV-SD7 strain（hereinafter referred to as SD7）were used as a poison to develop a bivalent inactivated vaccine for mink enteritis parvovirus.Firstly,SD1 and SD7were inoculated into CRFK cells to culture,then frozen and thawed three times to obtain toxicity.Both SD1 and SD7 cultures were titrated and diluted to 10^6.0TCID₅₀/mL as antigenic solution;SD1,SD7 and SD1+SD7 were added to formaldehyde at concentrations of 2‰,2.5‰,3‰,3.5‰at 37℃for 24 h,36 h,and 48 h,respectively.Inactivate the antigen solution at 0.5 mL/bottle.Single-layer CRFK cells were inoculated and passed blindly for 3passages.If no cell lesion（CPE）were produced,they were considered to be completely inactivated.As a result,the final concentration of formaldehyde was 3‰inactivated for 36 h.According to the ratio of 97:3,the inactivated antigen solution is mixed with Tween-80as the aqueous phase for preparing the vaccine;then the white oil adjuvant and the aqueous phase are emulsified according to a ratio of 2:1 to prepare an oil adjuvant.Inactivated vaccines were named Y-SD1,Y-SD7 and Y-SD1/SD7.The antigen-solution inactivated vaccine was prepared by mixing the antigen solution with Gel-01 adjuvant in a ratio of 1:1,and named G-SD1,G-SD7 and G-SD1/SD7,respectively.Appearance test,stability and dosage form test,sterility test and shelf life test were carried out on the prepared inactivated vaccines.As a result,the vaccine was clear and non-precipitating,free of microbial contamination,and stored at 4℃for at least 6 months.For safety testing,21 minks were randomly divided into 7 groups,and 1-6 groups were intramuscularly inoculated with Y-SD1,Y-SD7,Y-SD1/SD7,G-SD1,G-SD7 and G-SD1/SD7.The dose was 5 mL/only,and the 7th group was used as a control group,and an equal dose of physiological saline was intramuscularly injected.The experimental animals were observed for clinical symptoms,body temperature,and pathology.As a result,there was no abnormal clinical manifestation of mink and no pathological changes,indicating that the six vaccines were safe.The immune effects of the six inactivated vaccines were then tested.56 mink were randomly divided into 7 groups of 8 animals each.Groups 1-6 were inoculated with G-SD1,Y-SD1,G-SD7,Y-SD7,G-SD1/SD7 and Y-SD1/SD7 inactivated vaccines at a dose of 1.0mL/group,and group 7 served as a control injected the same dose of normal saline.The experimental animals were subjected to blood collection and serum preparation at the 7th,14th,21th,28th day after vaccination.The antibody titer of anti-MEV and FPV in serum was detected by HI test.The results of 28 d HI antibody test showed the average value of HI antibody in the serum of G-SD1 group was 1:1112,in the Y-SD1 group was1:936,in the G-SD7 group was 1:430,in the Y-SD7 group was 1:362,in the G-SD1/SD7 group is 1:1314,in the Y-SD1/SD7 group is 1:1217.It indicated that all the six vaccines can induce good antibody levels in the animal body,but the average antibody value of the bivalent inactivated vaccine group is larger than the average value of the antibody of the monovalent inactivated vaccine group;the vaccine group with different adjuvants of the same antigen solution,water adjuvant.The antibody average of the vaccine group was higher than that of the oil adjuvant vaccine group.After 30 days of vaccination,protective experiments were carried out.7groups of experimental mink were challenged by SD1 via oral route.The dose of 5 ml/only was used to observe the clinical symptoms,body temperature and pathology of 7 groups of mink.The results showed that G-SD1/SD7 group and Y-SD1/SD7 group had good mental state and no abnormal clinical manifestations;G-SD7 group（25%）,Y-SD7 group（37.5%）,G-SD1 group（12.5%）,Y-SD1 group（12.5%）,there were 2,3,1 and 1 mink with mild clinical symptoms such as mild diarrhea and decreased activity,but they were eventually tolerated.The control group showed typical clinical symptoms of mink virus,the incidence rate was100%,and the mortality rate was 75%.The clinical necropsy of the diseased mink was observed.The digestive system was observed to have obvious lesions.The PCR test was performed on the organs of the dead and the results showed all positive.The prepared 6 kinds of mink-intestinal parvovirus inactivated vaccines can provide immune protection to mink,but the bivalent inactivated vaccine is superior to the monovalent inactivated vaccine.This study shows that the bivalent inactivated vaccine of mink enteritis parvovirus can be used in the production of mink,preventing and controlling mink viral enteritis.